Elucidation of binding preferences of YEATS domains to site-specific acetylated nucleosome core particles

Acetylated lysine residues (Kac) in histones are recognized by epigenetic reader proteins, such as Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain-containing proteins. Human YEATS domains bind to the acetylated N-terminal tail of histone H3; however, their Kac-binding preferences at the level of the nucleosome are unknown. Through genetic code reprogramming, here, we established a nucleosome core particle (NCP) array containing histones that were acetylated at specific residues and used it to compare the Kac-binding preferences of human YEATS domains. We found that AF9-YEATS showed basal binding to the unmodified NCP and that it bound stronger to the NCP containing a single acetylation at one of K4, K9, K14, or K27 of H3, or to histone H4 multi-acetylated between K5 and K16. Crystal structures of AF9-YEATS in complex with an H4 peptide diacetylated either at K5/K8 or K8/K12 revealed that the aromatic cage of the YEATS domain recognized the acetylated K8 residue. Interestingly, E57 and D103 of AF9, both located outside of the aromatic cage, were shown to interact with acetylated K5 and K12 of H4, respectively, consistent with the increase in AF9-YEATS binding to the H4K8-acetylated NCP upon additional acetylation at K5 or K12. Finally, we show that a mutation of E57 to alanine in AF9-YEATS reduced the binding affinity for H4 multiacetylated NCPs containing H4K5ac. Our data suggest that the Kac-binding affinity of AF9-YEATS increases additively with the number of Kac in the histone tail.

Posttranslational modifications (PTMs), such as acetylation and methylation of lysine residues, occur in a wide variety of cellular proteins, including core histones (1)(2)(3). These PTMs of the N-terminal tails of the core histones H2A, H2B, H3, and H4 can be inherited across cell divisions through active maintenance by lysine acetyltransferases and methyltransferases and control chromatin structure and gene expression in eukaryotes (4)(5)(6). These PTMs are added and maintained in the nucleosome core particle (NCP), a chromatin compaction unit formed by wrapping the histone octamer (two copies of each of the four core histones) with a 145 to 147 bp dsDNA (7)(8)(9). In the NCP, the N-terminal tails of the core histones protrude through the DNA (7) and are accessible to a variety of chromatin-associated factors, such as 'writers', 'readers', and 'erasers' of the PTMs (5).
Lysine acetylation of H3 and H4 is a key regulator of gene expression; its effect can be direct or indirect. The direct effect is caused by the removal of the positive charges of lysine side chains by acetylation. Consequently, the acetylation of the N-terminal tail of H4 decreases its affinity to nucleosome-length DNA (10). Single acetylation of H4 at K16 in nucleosomes reduces the internucleosome interaction, and multiple acetylation of H4 at K5/K8/K12/K16 (i.e., hyperacetylation of H4) causes internucleosomal decompaction in reconstituted systems (11)(12)(13), suggesting that lysine acetylation in the nucleosome decompacts the higher-order chromatin structure even in the absence of chromatin-associated factors. The indirect effect of lysine acetylation on chromatin regulation is mediated by recruitment of a 'reader' domain (14,15). Acetylated lysine (Kac) in the N-terminal tail of H3 or H4 provides a binding scaffold for reader proteins such as those containing a bromodomain or Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain. The bromodomain was the first Kac-binding domain to be identified; it consists of approximately 110 residues and forms a four-α-helix bundle structure (16)(17)(18). In humans, 61 bromodomains are present in 46 different proteins (18). Another Kac-binding domain, the YEATS domain, consists of approximately 130 residues and forms an antiparallel β-sheet structure that can recognize and bind Kac and some acylated lysines (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). In humans, four YEATS domains are present in four different proteins (AF9, ENL, YEATS2, and GAS41) (29).
Lysine acetylation in the histone tail enhances the binding of the bromodomain and the YEATS domain, as demonstrated by biochemical analyses performed mostly with acetylated peptides as binding substrates (21,(26)(27)(28)(30)(31)(32), but rarely with acetylated nucleosomes (33). Arrays of histone tail peptides containing a variety of combinatorial PTMs, immobilized on a membrane, beads, or a plate, have been developed for such analyses (34)(35)(36). They are especially useful for validation of antibodies recognizing a PTM in a histone tail. However, it is better to validate the preference of chromatin-associated factors for PTMs by using nucleosomes with PTMs as substrates, because a histone peptide and a nucleosome differ chemically and physically, and the latter better reflects the chromatin environment.
Binding of YEATS domains to acetylated histones has been analyzed mostly using acetylated histone peptides; for example, binding of AF9-YEATS to an H3K9ac peptide (21) and binding of the YEATS domains of ENL, YEATS2, and GAS41 to an H3K27ac peptide (26-28, 31, 32, 37) have been analyzed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance. On the other hand, there is only one report that AF9-YEATS binds to unmodified and H3K9acetylated NCPs in a native gel shift assay (38). Colocalization of ENL, YEATS2, and GAS41 with H3K27ac in cells has been analyzed by ChIP-seq, co-IP, or both (26,27,31,32). However, the binding affinity and selectivity of each YEATS domain for binding to unmodified and specifically acetylated nucleosomes have not been determined.
Currently, nucleosomes with residue-specific acetylation(s) are prepared by native chemical ligation or by genetic code reprogramming. Studies on Kac incorporation into histones through genetic code reprogramming are still limited (39)(40)(41)(42). Chin et al. (42) reported biochemical preparation of histones H2A, H2B, and H3, each of which carried genetically installed Kac at a single defined residue. We have developed a biochemical methodology to synthesize a protein in which Kac, or a variety of lysine analogs, can be introduced at multiple positions through reprogramming of the genetic code reprogramming and engineering of the translation termination system (13,(43)(44)(45). In particular, utilization of a cell-free protein synthesis system enables milligram-scale production of the Kac-containing H4 protein (13). An NCP-containing H4 tetra-acetylated at K5/K8/K12/K16 produced by this methodology has essentially the same structure and stability as the unmodified NCP (13), demonstrating its structural integrity. However, the genetic preparation of a variety of Kac-histones and NCPs for comparative biochemical analyses has yet to be performed.
Here, we reconstituted NCPs containing a series of residuespecific acetyllysine(s) in the N-terminal tail of histone H3 or H4 for comparative validation of Kac-binding proteins. We prepared a mini library of Kac-containing NCPs immobilized on an avidin-coated multiwell plate and optimized the binding conditions using several bromodomain proteins. We quantitatively compared the Kac-binding preferences of four human YEATS domains toward acetylated nucleosome or acetylated tail peptides and found that AF9-YEATS binds to unmodified NCP and that AF9 and two other YEATS domains bind to the NCP containing H4 tetra-acetylated at K5/K8/K12/K16. To understand how the AF9-YEATS domain recognizes multiacetylation of H4, we solved its crystal structures in complex with H4 peptides diacetylated either at K5/K8 or K8/K12. In both structures, AF9 recognized K8ac at the aromatic cage of the YEATS domain, while residues outside the aromatic cage interacted with K5ac or K12ac. We discuss the structural mechanism of the recognition of H4 multiacetylation by AF9-YEATS and a potential advantage of the NCP library over the conventional histone peptide array in characterizing Kacbinding proteins.
First, we examined whether the reconstituted NCP with Kac is functional as a substrate for lysine deacetylases (KDACs) in vitro. We tested activity of histone deacetylase 6 (HDAC6) toward NCP-containing H4 with K5ac, K8ac, K12ac, or K16ac. Our dot blot analysis showed that HDAC6 deacetylated all four Kac residues (Fig. 1B). Subsequently, we examined whether a HDAC-specific inhibitor, trichostatin A (TSA), inhibits deacetylation of Kac-containing NCPs. TSA (1 μM) inhibited deacetylation of all four Kac residues by HDAC6 (Fig. 1B). HDAC6 seemed to prefer K12ac in a dose-dependent assay with NCPs acetylated at single H4 residues. These results indicate that reconstituted NCPs with residue-specific acetylation(s) are functional substrates for a KDAC and that this acetylation is completely or almost completely erasable by a KDAC in vitro.
Next, to compare the binding preferences of Kac-binding proteins at the nucleosome level, we immobilized 12 kinds of Kac-containing NCPs with biotinylated H2B onto a streptavidin-coated multiwell plate (Fig. 1C). Next, we optimized the conditions of in vitro association between the reconstituted NCP and Kac-binding proteins by using three human bromodomains with known binding specificities for acetylated histone tail peptides (Fig. 1D). To detect binding by fluorescence intensity, the second bromodomain (BD2) of PB1, bromodomain of BAZ2B, or double bromodomain (BD1+BD2) of TAF1L was fused to a green fluorescent protein, Azami-Green (AG) (47). The binding experiments were performed in 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl (Fig. 1D), which is considered a physiological saline condition. Under the washing and binding conditions shown in Figure 1D, the AG protein alone (negative control) did not bind to any of the NCPs (Fig. S1A). AG-fused PB1-BD2 significantly bound to the H3K14-acetylated NCP in comparison with the unmodified NCP (p < 0.01; Fig. S1A), as expected from previous NMR and biochemical binding analyses (30,48). BAZ2B bromodomain also preferentially bound to the H3K14-acetylated NCP (p < 0.01; Fig. S1A), which is consistent with previous reports (30,49). The TAF1L bromodomain binds weakly to H3 or H4 acetylated at various residues, such as H3K4ac and H4K5ac (30). In the NCP array, TAF1L bound to many kinds of acetylated NCPs (Fig. S1A). Because the binding preferences obtained in this assay were consistent with previous reports, we concluded that the acetylated NCP-based assay established in this study is valid for evaluating the interactions of epigenetic reader proteins with posttranslationally modified nucleosomes. The binding preferences of other typical bromodomains were determined under the same assay conditions (Fig. S1B).

Analysis of binding between the acetylated NCP array and YEATS domains
The domain architecture of the YEATS domain-containing proteins is shown in Figure 2A. We purified AG-fused YEATS domains of AF9, ENL, YEATS2, and GAS41 and measured their binding to acetylated NCPs (Fig. 2B). In comparison with binding to the unmodified NCP, the AF9-YEATS domain bound strongly and significantly to the H3K9-acetylated NCP (p < 0.01), as expected from (21); it also bound significantly to the H3K4-and H3K27-acetylated NCPs (p < 0.01). It bound weakly to all other NCPs including the unmodified one. The ENL-YEATS domain bound significantly to the H3K4-, H3K9-, and H3K27-acetylated NCPs (p < 0.05) and also showed basal-level binding to all other NCPs, but it was weaker than that of AF9-YEATS.
The YEATS2-YEATS domain reportedly prefers H3K27ac (31) and binds to K27ac-containing H3 peptides with a K D of 226 μM (50) or 50 μM (31). In our assay, it did not show significant interaction with any of the acetylated NCPs, although it slightly preferred H3K27ac (Fig. 2B). Using ITC analysis, we also found that YEATS2-YEATS protein did not bind to the K27ac-containing H3 (15-39) peptide (Fig. S2). To confirm that our YEATS2-YEATS protein was active, we examined its binding to K27-crotonylated H3 (1-34) peptide, to which YEATS2-YEATS reportedly binds stronger (K D = 31.7 μM) than to the K27ac-containing H3 peptide (50). In our ITC analysis, YEATS2-YEATS bound to the K27cr-containing H3  peptide with a K D of 340 μM (Fig. S2), indicating that it was active. We also confirmed that the fusion of AG to YEATS2-YEATS had little effect on this binding (Fig. S2). Together, these results suggest that the active YEATS2-YEATS domain used in this study does not significantly prefer any acetylated NCPs over the unmodified NCP.
The GAS41-YEATS domain significantly bound to H3K4-, K9-, K14-and K27-acetylated NCPs (p < 0.05; Fig. 2B); the binding was strongest to H3K27ac. Statistical significance of YEATS domain binding to Kac is summarized in Figure 2C. We also found that the YEATS domains of AF9, ENL, and GAS41 bound significantly to H4K5/K8/K12/K16-tetra-acetylated NCP (Fig. 2, B and C). Next, we compared the Kac-binding preferences of YEATS domains to acetylated NCPs and to acetylated histone tail peptides. Binding results obtained using a MODified Histone Peptide Array are shown in Fig. S3. YEATS domains of AF9 and GAS41 reproducibly bound to the histone H3 tail peptide containing K27ac. YEATS2-YEATS bound weakly and nonreproducibly to H3K27ac. These results were consistent with recent reports (21,31,32). The YEATS domains of AF9, ENL, and GAS41 bound to the H4 (1-19) tail peptide when it was diacetylated at K5/K8 or tetra-acetylated at K5/K8/K12/K16. The YEATS domains of all four proteins scarcely bound to any of the unmodified histone tail peptides (i.e., H2A, H2B, H3, or H4; Fig. S3).

The AF9-YEATS domain binds to the unmodified NCP
We measured the K 1/2 (the half-saturation concentration for NCP binding) of the interaction between AF9-YEATS and the unmodified NCP or 147-bp dsDNA using microscale thermophoresis. The K 1/2 value of AF9-YEATS binding to the unmodified NCP composed of 147-bp dsDNA and the histone octamer (0.14 ± 0.01 μM) was 86 times lower (i.e., the binding was stronger) than that for nucleosome-free 147-bp dsDNA (12 ± 0.1 μM; Fig. 3A).
to nucleosome-free 147-bp dsDNA (8.2 ± 0.6 μM; Fig. 3A) indicated that the interaction between YEATS2-YEATS and the unmodified NCP is detectable by microscale thermophoresis. The respective K 1/2 values for GAS41-YEATS (1.4 ± 0.1 and >50 μM; Fig. 3A) also indicated that the interaction between GAS41-YEATS and the unmodified NCP is detectable by microscale thermophoresis. Thus, the binding affinity of YEATS2-YEATS to nucleosome-free dsDNA was similar to that of AF9, whereas that of GAS41-YEATS was much weaker. The results, together with the binding results obtained using the MODified Histone Peptide Array, are summarized in Figure 3B.

Surface potentials of the YEATS domains
The ability of YEATS domains to bind the NCP and nucleosome-free dsDNA is assumed to be regulated by their electrostatic surface potentials (38). The surface potential of AF9-YEATS (PDB ID: 4TMP) is shown in Figure 3C. Three major positively charged surface regions in AF9-YEATS are composed of 1) R61, K63, and R64; 2) K92, R96, and K97; and 3) R133, K134, and K137.
To investigate the folding and electrostatic potential of YEATS2-YEATS, we determined the crystal structure of the YEATS2-YEATS apo-form at 1.67 Å resolution. The overall structure was similar to that of AF9-YEATS with an alpha carbon RMSD value of 0.76 Å, indicating the absence of misfolding in our YEATS2-YEATS protein. The electrostatic surface potential of YEATS2-YEATS is shown in Figure 3C. Similar to AF9-YEATS, YEATS2-YEATS had three major positively charged surface regions: 1) K217, K233, and R280; 2) K239, K242, and K243; and 3) K253, R271, and R300.
The surface of GAS41-YEATS (PDB ID: 5XTZ) was more negatively charged than those of AF9-YEATS or YEATS2-YEATS (Fig. 3C), which was consistent with dsDNA binding to AF9-YEATS and YEATS2-YEATS, but not to GAS41-YEATS.
Molecule A in complex with the H4K5ac/K8ac peptide (chain A) and molecules A and B in complex with two H4K8ac/K12ac peptides (chains A and B) recognized H4K8ac at the aromatic cage (Figs. 5B, S4, A and B). Molecule B in the H4K5ac/K8ac peptide complex did not bind the peptide at the aromatic cage because the aromatic cage was closed by conformational changes around Y78 in AF9 (Fig. S4C). The positively charged R61 and R64 residues of molecule B hydrogen bonded with chain A in both complexes (Fig. S4, A  and B). The electron density of H4K5ac was detected in the H4K5ac/K8ac peptide complex structure, but the position of the side chain of unmodified H4K5 in the H4K8ac/K12ac peptide complex could not be determined because of low electron density (Fig. 5A). K5ac and K12ac were located outside the aromatic cage; Nε of H4K5ac hydrogen bonded with E57 (Fig. 5B, left) and the added acetyl group of H4K12ac hydrogen bonded with D103 via a water molecule (Fig. 5B,  right). Outside this cage, the main chain of unmodified H4K12 in the H4K5ac/K8ac peptide complex also hydrogen bonded with D103 via two water molecules, whereas its side chain was oriented toward the solvent (Fig. 5B, left). Two-dimensional plots of residues interacting with the H4K5ac/K8ac or H4K8ac/K12ac peptide are shown in Figure 5C. The added acetyl group of H4K12ac interacted hydrophobically with H116 of AF9. These structures indicate that the aromatic cage of AF9-YEATS recognizes a single Kac (H4K8ac) even when the nearby lysines in the H4 N-terminal tail are acetylated.
The interaction modes of AF9-YEATS with H4K8ac and with H3K9ac (21) were the same: the aromatic cage of AF9-YEATS hydrophobically interacted with the aromatic residues F28, H56, F59, Y78, and F81; and the amide and the carbonyl oxygen of the Kac side chain formed hydrogen bonds with the hydroxyl group of S58 and the backbone amide of Y78 (Fig. 5B).
Binding of the AF9-YEATS domain to acetyllysine outside the aromatic cage Finally, we investigated whether the AF9-YEATS residues that interacted with the Kac adjacent to K8ac of the H4 peptide Binding preferences of YEATS to acetylated nucleosomes in the crystal are important for the interaction in solution. To this end, we replaced H4K5ac-interacting E57 or H4K12acinteracting D103 of AF9-YEATS with alanine and used the mutated proteins and multiacetylated NCPs in microscale thermophoresis (Fig. 6). The interaction was weaker for the E57A mutant than for the WT with any of the multiacetylated NCPs tested. The increase in K 1/2 toward K5/K8-diacetylated and tetra-acetylated NCPs was 1.8-and 1.5-fold, respectively. On the other hand, the increase of K 1/2 toward K8/K12diacetylated NCPs was 1.3-fold, which is the lowest among Binding preferences of YEATS to acetylated nucleosomes the three multiacetylated NCPs. This result suggests that the E57 residue is important for the interaction with multiacetylated NCPs containing H4K5ac. The D103A mutant did not interact with any of the multiacetylated NCPs, suggesting that this mutation affected protein folding.

Binding of multiacetylated H3 peptides by the AF9-YEATS domain
Intriguingly, Taf14 has higher affinity for multiacetylated histone H3 than for the monoacetylated one (51). To investigate whether AF9-YEATS preferentially binds to multiacetylated H3 and H4, we used ITC to measure its binding affinity toward five monoacetylated or diacetylated H3 (1-19) peptides (Fig. S5). Among peptides monoacetylated at K4, K9, or K14, AF9-YEATS specifically bound to the K9acetylated H3 peptide with a K D of 7.0 μM. Additional acetylation at K4 slightly increased its affinity (K D = 5.8 μM), whereas that at K14 slightly decreased it (K D = 8.5 μM). These results suggest that, similar to the role of K5ac in the K8acmediated interaction of the H4 tail peptide, K4ac in the H3 tail peptide contributes to the K9ac-mediated interaction outside of the YEATS domain cavity.

Discussion
Identification of the preferences of chromatin-associated factors for PTMs is important in understanding their action and mechanism. In a few available reports on the development of PTM-containing nucleosome libraries, these libraries were prepared by native chemical ligation, that is, by conjugating a PTM-containing histone tail peptide with the histone core regions (33,52). Through genetic code reprogramming and biochemical validation using HDAC6 (Fig. 1) and bromodomains (Fig. S1), we established an NCP-based assay platform and used it to compare the Kac-binding preferences of YEATS domains at the histone peptide and nucleosome levels (Fig. 2). Theoretically, the methodology developed here is applicable not only to histones and the NCP but to any PTM-containing proteins of interest. Because histones are composed of an unfolded N-terminal tail region and a folded C-terminal core region, they are ideal models for the native chemical ligation method. Our methodology may be advantageous over native chemical ligation when applied to the position-specific introduction of Kac into a folded region of a protein other than a histone. Several pyrrolysyl-tRNA synthetase mutants introduce crotonylated lysine site specifically (53, 54), so it is theoretically possible to prepare a nucleosome containing a site specifically crotonylated histone. The crotonylated histone is reportedly recognized by the YEATS domain and regulates transcription (24). Using the NCP-based assay, it would be possible to apply it to a variety of epigenetic reader proteins to investigate their binding properties to crotonylated nucleosomes that were not revealed by the histone peptide-based assay.
Because the histone peptides in the peptide array are unfolded, the YEATS domain can access all the acetylated residues in histone peptides without any steric hindrance, whereas histone tails protrude from the structured NCP and the access of the YEATS domain to the acetylated histone tails may be sterically hindered by the NCP. Thus, the NCP-based binding assay better reflects the chromatin environment in the nucleus than the peptide-based binding assay does; the former assay excludes artifactual binding. Although YEATS2-YEATS reportedly binds a K27ac-containing H3 peptide (31), our YEATS2-YEATS protein scarcely bound to an NCP containing H3K27ac and did not significantly prefer it over the unmodified NCP (Fig. 2, B and C). In our ITC assay, YEATS2-YEATS did not bind to the K27-acetylated H3 peptide but bound to the K27-crotonylated H3 peptide (Fig. S2). Peptide array assay showed that YEATS2-YEATS weakly and nonreproducibly bound to the K27-acetylated H3 peptide (Fig. S3). Hence, although we cannot exclude the interaction between YEATS2-YEATS and H3K27ac at the peptide level, our NCP-based binding assay revealed that YEATS2-YEATS did not prefer any of the examined H3/H4 acetylations.
This assay is also useful for detecting and evaluating binders that interact with DNA or histones but not with the PTM. In particular, this study suggests that different YEATS domains interact with different sets of components (dsDNA, histones, or both) of the unmodified NCP (Fig. 3B). Using fluorescence spectroscopy assay, Klein et al. (38) have found that AF9-YEATS may bind to the unmodified NCP owing to its DNAbinding ability: it binds to 15-bp dsDNA (K D = 47 μM) and to 20-bp dsDNA (K D = 57 μM). In our microscale thermophoresis assay (Fig. 3, A and B), AF9-YEATS bound to 147-bp dsDNA and 86 times stronger to the unmodified NCP (Fig. 3A). These results suggest that AF9-YEATS interacts with the unmodified NCP through DNA and also substantially through histones (Fig. 3B). YEATS2-YEATS bound much weaker to the unmodified NCP than AF9-YEATS did, but it bound to 147-bp dsDNA slightly stronger than AF9-YEATS did. On the electrostatic potential maps, AF9-YEATS had no apparent positively charged surface region near the Kac-binding site, whereas YEATS2-YEATS had several positively charged residues, such as K217, K233, and R280 (Fig. 3C); these positively charged residues may interact with dsDNA and thus enhance the dsDNA-binding ability of YEATS2-YEATS. The affinity ratio of YEATS2-YEATS toward the unmodified NCP versus 147-bp dsDNA was small (3.2). Given these binding properties, YEATS2-YEATS may interact quite weakly with histones (Fig. 3B).
GAS41-YEATS may interact with the unmodified NCP through histones because it had considerable affinity to the unmodified NCP whereas it bound weakly to 147-bp dsDNA (Fig. 3B). Overall, the binding affinity of YEATS domains for the unmodified NCP cannot be simply explained by their interaction with DNA, and interaction with the histones within the NCP presumably matters (Fig. 3B). We attempted to identify the residue(s) of AF9-YEATS that interact(s) with the unmodified NCPs by NMR measurements using the transferred cross-saturation technique, cocrystal structure analysis, and cryo-EM. However, these experiments did not uncover the mechanism of AF9-YEATS binding to the unmodified NCP because of problems with sample preparation.
We solved the crystal structures of AF9-YEATS complexed with the K5/K8-and K8/K12-diacetylated H4 peptides (Fig. 5A). In these asymmetric units, two AF9-YEATS molecules seemingly interacted with each other via positively charged residues, such as R61 and R64, and the H4 peptide (Fig. S4, A and B). However, AF9 is monomeric in solution (21), and these positively charged residues are responsible for DNA binding (38). Because the AF9-YEATS domain cannot dimerize in solution and the interaction between the asymmetric AF9-YEATS molecule and the H4 peptide competes with the reported DNA binding, the H4 peptide-binding mode of the AF9-YEATS dimer observed in the crystal is probably caused by crystal packing and may not occur in solution. Thus, the increased affinity to multiple Kac is not due to the dimerization of AF9-YEATS and is presumably attributable to the recognition between a single aromatic cage of AF9-YEATS and a single Kac, accompanied by additional recognition of another nearby Kac within the same histone tail by the residues (i.e., E57 and D103) located outside the aromatic cage.
Intriguingly, when another Kac was present near K8ac in the histone H4 tail (e.g., K5ac), it interacted with AF9-YEATS (Fig. 4) at the surface outside of the aromatic cage of the YEATS domain in the crystal (Fig. 5, B and C). Our biochemical analysis indicates that, at least via E57, AF9-YEATS senses K5ac adjacent to K8ac in the H4 tail, which additively increases the affinity toward the NCP (Fig. 6). Similarly, AF9-YEATS may also sense K4ac adjacent to K9ac in the H3 tail (Fig. S5). Hence, the simultaneous presence of two or more Kac in the H3 and/or H4 tail may increase the number of such interactions using both the YEATS domain cavity and its outer surface.
Another Kac-binding domain, the N-terminal bromodomain of the members of the bromodomain and extra-terminal domain family, can bind to the K5/K8-diacetylated histone H4 tail through simultaneous recognition of two Kac in the same bromodomain cavity (Fig. S4D) (30,55). Intriguingly, the mode of K5/K8 diacetylated H4 recognition by the AF9-YEATS domain was completely different from that by the bromodomain of the bromodomain and extra-terminal domain family. The AF9-YEATS domain primarily recognized H4K8ac regardless of acetylation at K5 or K12. Importantly, newly synthesized H4 is diacetylated in the cytoplasm at K5 and K12, and other lysine residues in the N-terminal tail, including K8, can be further acetylated in the nucleus (56,57). Therefore, the mode of primary binding to K8ac and secondary binding to K5ac or K12ac by the AF9-YEATS domain provides a unique molecular basis for sensing the hyperacetylation of H4 in the nucleus. Approaches such as coimmunoprecipitation and ChIP-seq are needed to establish whether the direct binding of AF9 to the H4 multiacetylated NCP is involved in the regulation of a nuclear function such as gene expression, as in the case of AF9 binding to H3K9ac (21).
In conclusion, we established a binding assay based on site specifically acetylated NCPs and found that the AF9-YEATS domain binds to H4-multiacetylated NCPs. The crystal structures of the AF9-YEATS domain in complex with H4 peptides revealed that K8ac is recognized within the aromatic cage of the YEATS domain, whereas the nearby K5ac and K12ac contribute to the binding outside of the aromatic cage. These structural and mutagenesis analyses provide a binding model in which the Kac-binding affinity of AF9-YEATS increases additively with the number of Kac in the histone tail. Our analyses suggest that the YEATS domain of AF9 binds the NCP mainly through DNA and histones, that of YEATS2 binds mainly through DNA, and that of GAS41 binds mainly through histones. The methodology presented here will enable preparation of a library of nucleosomal beads-on-a-string. Characterization of chromatin-associated factors in the context of higher-order chromatin with PTMs will aid in understanding their functions in the natural chromatin environment in the nucleus.

Reconstitution of H2B-biotinylated NCPs
The H3.1-or H4-acetylated NCPs along with the unmodified NCPs were reconstituted as described (13,59), using Kac-Binding preferences of YEATS to acetylated nucleosomes containing H3.1 or H4, unmodified histones, biotinylated H2B, and 147-bp palindromic human α-satellite DNA. Briefly, purified histones were mixed at an equimolar ratio in a solution containing 7 M guanidine-hydrochloride and 10 mM DTT and dialyzed against 10 mM Tris-HCl buffer (pH 7.5) containing 2 M KCl and 5 mM DTT at 4 C for 4 h; dialysis buffer was exchanged 4 times. Histone octamers were concentrated in Amicon Ultra-15 centrifugal units (Merck Millipore, MWCO 30 kDa) and purified by gel filtration chromatography using a HiLoad 16/60 Superdex 200 column (GE Healthcare). Histone octamers and 147-bp DNA were mixed at a 1.0 : 1.1 M ratio in 10 mM Tris-HCl buffer (pH 7.5) containing 2 M KCl, 1 mM EDTA, and 1 mM DTT. The solution was placed in a dialysis membrane bag (Spectrum, MWCO 6-8 kDa, cat. no. 132653) and dialyzed against the same buffer at 4 C for 4 h. The concentration of KCl was then gradually decreased by diluting for 30 h with 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA and 1 mM DTT using a peristaltic pump. The reconstituted NCP samples were incubated at 55 C for 2 h. To separate free DNA, MgCl 2 was added to the reconstituted NCP samples at a final concentration of 12 mM. The NCPs were precipitated by centrifugation at 17,500g for 10 min at 4 C and suspended in 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA and 1 mM DTT.

Nucleosome deacetylation and deacetylation inhibition assays
The NCPs for deacetylation assays were reconstituted using unmodified H2B. In each deacetylation reaction, 200 ng of the Kac-containing NCP was incubated with 50, 100, 200, or 400 ng of HDAC6 (SignalChem; cat. no. H88-30 G) in 20 μl at 37 C for 16 h. In the inhibitor assays, TSA was added to the reaction mixture not containing NCP to a final concentration of 1 μM and preincubated at 37 C for 1 h; NCP monoacetylated either at K5, K8, K12, or K16 of H4 was then added. After the reactions, samples were spotted on a nitrocellulose membrane (BioTrace NT Nitrocellulose Transfer Membrane, 0.2 μm, Pall Corporation, cat. no. 66485) and immunoblotted using H4-specific antibodies, each recognizing acetylation at K5, K8, K12, or K16.

Preparation of bromodomains and YEATS domains
cDNAs-encoding bromodomains or YEATS domains were fused with a histidine-tagged sequence containing a SUMO protease digestion site at their N-termini and a full-length AG cDNA (47)   . A T7 promoter sequence was inserted at the 5 0 ends of the AG-fused protein expression constructs, and the cDNA cassettes were inserted into a pCR2.1 TOPO plasmid by using a TA cloning kit (ThermoFisher Scientific, cat. no. 450641). The AG-fused proteins were produced in cell-free protein synthesis reactions in 6 ml with 2 μg/ml template DNA, essentially as previously described (60). Then, the reaction mixtures were centrifuged at 28,000g for 20 min at 4 C and the AG-fused proteins were purified from the supernatants by HisTrap HP column chromatography (GE Healthcare). The columns were washed with 10 mM Tris-HCl buffer (pH 8.0) containing 1 M NaCl and 20 mM imidazole, and bound proteins were eluted with 10 mM Tris-HCl buffer (pH 8.0) containing 500 mM NaCl and 500 mM imidazole. Eluted fractions were dialyzed against 10 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl and 1 mM DTT and used for binding assay.
For the assay using the MODified Histone Peptide Array, the same cDNA sequences encoding human YEATS domains were amplified by PCR and subcloned into a pCR2.1 vector with a GST-encoding sequence inserted after an N-terminal polyhistidine tag. For microscale thermophoresis measurements, a similar construct but without GST was used. Sitedirected mutagenesis of AF9-YEATS (residues 1-138) was conducted by PCR using the DpnI restriction enzyme and the following primer sets: 5 0 -CCT TCA CGC ATC CTT TCC TCG TCC TAA AC-3 0 (forward) and 5 0 -GGA AAG GAT GCG TGA AGG TGA AAC ACC AC-3 0 (reverse) for E57A and 5 0 -CGA CTA TGA CCT GTT TCT GCA TCT CG-3 0 (forward) and 5 0 -GCA GAA ACA GTG CAT AGT CGA AGC GAA C-3 0 (reverse) for D103A. The introduced mutations were verified by DNA sequencing. For X-ray structural analysis, the cDNAs encoding AF9-YEATS (residues 1-138) and YEATS2-YEATS (202-338) were amplified by PCR and subcloned into the pCR2.1 vector encoding GST and the polyhistidine tag. For ITC experiments, the cDNAs encoding AF9-YEATS (residues 1-138) and YEATS2-YEATS (200-349) were inserted into the same vector as for X-ray analysis. The expression using this vector has been previously described (61). All YEATS domain proteins were purified on a HisTrap HP column (GE Healthcare), and the eluted fractions were loaded on a HiLoad Superdex 200 16/60 column (GE Healthcare) equilibrated with Tris-HCl buffer (pH 7.6) containing 500 mM NaCl and 1 mM DTT.

NCP-based binding assay
The surface of a Pierce streptavidin-coated high-capacity 96-well black plate (ThermoFisher Scientific, cat. no. 15503) was washed 3 times with 10 mM Tris-HCl buffer A (pH 7.5) containing 150 mM NaCl and 0.05% Tween-20. In each well, 5 pmol of the unmodified or acetylated NCPs containing biotinylated H2B were immobilized at 4 C for 16 h, NCP solution was removed, and the wells were washed 3 times with buffer B (10 mM Tris-HCl, pH 7.5, containing 150 mM NaCl). Then, buffer B containing 0.5% nonfat dried milk was added to each well and was incubated at 25 C for 1 h, and the wells were washed 3 times with buffer B. AG-fused bromodomain or YEATS domain proteins (25 pmol each) were added to each well, incubated at 25 C for 1 h, and the wells were washed 3 times with buffer B. Fluorescence intensity was measured in an EnVision multilabel plate reader (PerkinElmer) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The binding ratio was calculated for each well by dividing fluorescence intensity after the final wash (bound fraction) by the initial fluorescence intensity at the addition of AG-fused protein. All binding experiments were performed in triplicate. A two-tailed Student's t test was performed for statistical analysis of preference for acetylated versus unmodified NCPs. We classified p value <0.05 as *, p value <0.01 as **.

Peptide array-based binding assay
Assays were performed using the MODified Histone Peptide Array (Active Motif) following the manufacturer's protocol. Briefly, the arrays were blocked by incubation in 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.05% Tween-20, and 5% nonfat dried milk at 4 C overnight. The arrays were then washed with 50 mM Tris-HCl (pH 7.6) containing 250 mM NaCl, 0.1% NP-40, and 10% glycerol and incubated with the 10 μM YEATS domain at 4 C overnight with gentle agitation. Arrays were washed with 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.05% Tween-20 and incubated with anti-GST primary antibodies at 4 C overnight (1:2000 dilution) and then with anti-rabbit HRP secondary antibodies (1:2500 dilution). Protein binding was detected by Chemi-Lumi One Super (Nacalai Tesque). Signal intensities were quantified by using the Protein Array Analyzer for ImageJ (written by Gilles Carpentier, 2010. The macro is available online: http://rsb.info. nih.gov/ij/macros/toolsets/Protein Array Analyzer.txt).

Isothermal titration calorimetry
Measurements were performed at 15 C in 20 mM Tris-HCl (pH 7.6) containing 500 mM NaCl on a MicroCal Auto-iTC200 microcalorimeter (Malvern). Protein (100 μM, ca. 400 μl) was loaded into the sample cell, and acetylated peptides (1 mM) were loaded into the injection syringe. The collected data were analyzed using Origin 7 SR4, ver. 7.0552 software (OriginLab Corporation) supplied with the instrument to yield enthalpies of binding (ΔH) and K D . In all cases, a single binding site model was used.

Crystallization and structural analysis
Crystallization was performed by the sitting-drop vapordiffusion method at 20 C by mixing equal volumes of the YEATS domain of AF9 or YEATS2 (each at 7 mg/ml) and a reservoir solution. Crystals of the AF9-YEATS domain complexed with the H4K5ac/K8ac peptide were grown in 100 mM Bis-Tris buffer (pH 6.5) containing 100 mM ammonium acetate and 25% PEG 3350. Crystals of the AF9-YEATS domain complexed with the H4K8ac/K12ac peptide were grown in 200 mM ammonium citrate tribasic buffer (pH 7.0) containing 19% PEG3350. Crystals of the apo-form YEATS2-YEATS domain were grown in 100 mM Tris-HCl buffer (pH 8.5) containing 2 M NaCl. The crystals were briefly soaked in a cryoprotectant drop composed of the reservoir solution supplemented with 20% glycerol and then flash-cooled in liquid nitrogen for X-ray diffraction data collection. The datasets were collected at the beamline BL26B2, SPring-8 (Harima), and were processed with XDS and HKL2000 (62,63). Crystal structures were determined by molecular replacement using MOLREP with the structure of AF9 (PDB ID: 4TMP) as the search model. Model building was accomplished with Coot (64,65), and structural refinement was performed with REFMAC and PHENIX (66,67). Two-dimensional interaction plots were drawn with LIGPLOT (68). The structural models in the figures were drawn using PyMOL software (Schrodinger, LLC). All electrostatic surface maps were calculated using the APBS tool in a range of −5 kT e −1 to +5 kT e −1 (69).
Supporting information-This article contains supporting information.